Genetic and Functional Characterization of Isolated Stromal Cell Lines from the Aorta-Gonado-Mesonephros Region

ArticleinAnnals of the New York Academy of Sciences 1044(1):51-9 · July 2005with11 Reads
DOI: 10.1196/annals.1349.007 · Source: PubMed
The hematopoietic system interacts with a supportive stromal environment allowing maintenance and differentiation of hematopoietic stem cells (HSCs). The aorta-gonado-mesonephros (AGM) region serves as a unique embryonic microenvironment, generating the first adult repopulating HSCs in the mouse embryo. To eludicate factors involved in hematopoietic support and induction of hematopoietic differentiation, we isolated more than 100 stromal cell clones derived from the AGM region of embryonic day (E) 10.5 mouse embryos for functional and genetic analysis. Selected isolated AGM stromal cell lines are highly efficient in supporting maintenance and expansion of mouse and human hematopoietic stem and progenitor cells. In addition, we can demonstrate for the first time that AGM stromal cell lines are also potent inducers of hematopoietic differentiation of murine embryonic stem cells. Stromal gene array analysis has identified genes that could play a role in hematopoietic support.
  • [Show abstract] [Hide abstract] ABSTRACT: In all vertebrate animals, the first blood and vascular endothelial cells are formed during gastrulation, a process in which the mesoderm of the embryo is induced and then patterned by molecules whose identity is still largely unknown. Clusters of developing blood cells surrounded by a layer of endothelial cells comprise the "blood islands" and form in the visceral yolk sac, external to the developing embryo proper. Despite the identification of genes, such as Flk1, SCL/tal-1, Cbfa2/Runx1/AML1, and CD34, that are expressed during the induction of primitive hematopoiesis and vasculogenesis, the early molecular and cellular events involved in these processes are not well understood. Recent work has demonstrated that extracellular signals secreted by a layer of visceral endoderm surrounding the embryo are essential for the initiation of these events. A member of the Hedgehog family of signaling molecules is produced by visceral endoderm and is required for formation of blood and endothelial cells in explant cultures. Hedgehog proteins also stimulate proliferation of definitive hematopoietic stem/progenitor cells. Therefore, these findings may have important medical implications for regulating hematopoiesis and vascular development for therapeutic purposes and for the development of new sources of hematopoietic stem cells for transplantation and as targets for gene therapy.
    Article · Nov 2001
  • [Show abstract] [Hide abstract] ABSTRACT: During mammalian embryonic development the definitive haematopoietic stem cells (HSCs) may arise either in the extra-embryonic mesoderm or in the aorta-gonad-mesonephros (AGM) region that forms in close proximity to the assembling urogenital system, generating the gonad, cortex of the adrenal gland and metanephros. Researchers have been attempting for a long time to define the region of importance for generating the definitive HSCs that colonize the fetal liver and bone marrow, the two major sites where haematopoiesis takes place in the adult. The fetal liver might gain HSCs from both of the primary haematopoietic sources, but the extra-embryonic HSCs seem not to be able to colonize adult bone marrow directly. It is known that the microenvironment around the HSCs is important for directing cell fates, but we do not yet have much idea about the cell-cell interactions, tissue interactions and molecules that regulate cell behaviour in the AGM. We will here discuss the contribution of the AGM to definitive haematopoiesis in mammals and review some of the cell-cell interactions and associated signalling systems involved in the development of AGM stem cells.
    Article · Nov 2005
  • [Show abstract] [Hide abstract] ABSTRACT: Hematopoietic stem cells (HSCs) give rise to all blood and immune cells and are used in clinical transplantation protocols to treat a wide variety of refractory diseases, but the amplification of HSCs has been difficult to achieve in vitro. In the present study, the expansive effects of aorta-gonad-mesonephros (AGM) region derived stromal cells on HSCs were explored, attempting to improve the efficiency of HSC transplantation in clinical practice. The murine stromal cells were isolated from the AGM region of 12 days postcoitum (dpc) murine embryos and bone marrow (BM) of 6 weeks old mice, respectively. After identification with flow cytometry and immunocytochemistry, the stromal cells were co-cultured with ESCs-derived, cytokines-induced HSCs. The maintenance and expansion of ESCs-derived HSCs were evaluated by detecting the population of CD34+ and CD34+Sca-1+ cells with flow cytometry and the blast colony-forming cells (BL-CFCs), high proliferative potential colony-forming cells (HPP-CFCs) by using semi-solid medium colonial culture. Finally, the homing and hematopoietic reconstruction abilities of HSCs were evaluated using a murine model of HSC transplantation in vivo. AGM and BM-derived stromal cells were morphologically and phenotypically similar, and had the features of stromal cells. When co-cultured with AGM or BM stromal cells, more primitive progenitor cells (HPP-CFCs) could be detected in ESCs derived hematopoietic precursor cells, but BL-CFC's expansion could be detected only when co-cultured with AGM-derived stromal cells. The population of CD34+ hematopoietic stem/progenitor cells were expanded 3 times, but no significant expansion in the population of CD34+Sca-1+ cells was noted when co-cultured with BM stromal cells. While both CD34+ hematopoietic stem/progenitor cells and CD34+Sca-1+ cells were expanded 4 to 5 times respectively when co-cultured with AGM stromal cells. AGM region-derived stromal cells, like BM-derived stromal cells, could promote hematopoietic reconstruction and HSCs' homing to BM in vivo. AGM-derived stromal cells in comparison with the BM-derived stromal cells could not only support the expansion of HSCs but also maintain the self-renewal and multi-lineage differentiation more effectively. They are promising in HSC transplantation.
    Full-text · Article · Jan 2006
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